This application claims priority under 35 U.S.C. 119 of Tunisian application no. 99.100 filed on May 26, 1999, the contents of which are fully incorporated herein by reference.
1. Field of the Invention
The present invention relates to isolated polypeptides having glucose isomerase activity and isolated nucleic acid sequences encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides.
2. Description of the Related Art
D-xylose isomerase (D-xylose ketol isomerase, EC 5.3.1.5) catalyzes the conversion of D-xylose to D-xylulose in the first step of xylose metabolism following the pentose phosphate cycle, and of D-glucose into D-fructose as well, hence the enzyme is often referred to as glucose isomerase (GI) [Takasaki et al., 1969, Agric. Biol. Chem. 33: 1527-1534; Jensen V J. and et Rugh, S., 1987, Methods of Enzymol. 136: 256-370]. It is a key enzyme used for the production of sweet high fructose syrups that are used as alternative sweeteners to sucrose or invert sugar in the food and beverage industry.
Most food grade glucose is provided as an enzymatic hydrolysate of corn starch, i.e., corn syrup of commerce. Glucose is generally considered to be 60 to 80% as sweet as sucrose. It has long been known to isomerize glucose to fructose (which is even sweeter than sucrose) employing an enzyme having glucose isomerase activity. This may be a glucose isomerase which has been immobilized upon an inert support such as diethylaminoethyl-cellulose, porous glass or chitin or it can be cells that express glucose isomerase which have been immobilised by cross-linking, e.g. with glutaraldehyde . Detailed descriptions of the enzymatic conversion of glucose to fructose employing glucose isomerase can be found in Hamilton, et al. xe2x80x9cGlucose Isomerase a Case Study of Enzyme-Catalysed Process Technologyxe2x80x9d, Immobilized Enzymes in Food and Microbial Processes, Olson et al., Plenum Press, N.Y., (1974), pp. 94-106, 112, 115-137; Antrim, et al., xe2x80x9cGlucose Isomerase Production of High-Fructose Syrupsxe2x80x9d, Applied Biochemistry and Bioenqineering, Vol. 2, Academic Press (1979); Chen, et al., xe2x80x9cGlucose Isomerase (a Review)xe2x80x9d, Process Biochem., (1980), pp. 30-35; Chen, et al. xe2x80x9cGlucose Isomerase (a Review)xe2x80x9d, Process Biochem., (1980), pp. 36-41; Nordahl, et al., xe2x80x9cFructose Manufacture from Glucose by Immobiled Glucose Isomerasexe2x80x9d, Chem. Abstracts, Vol. 82, (1975), Abs. No. 110316h; and Takasaki, xe2x80x9cFructose Production Glucose Isomerasexe2x80x9d, Chem. Abstracts, Vol. 82, (1975), Abs. No.110316h; and Takasaki, xe2x80x9cFructose Production by Glucose Isomerasexe2x80x9d, Chem. Abstracts, Vol. 81, (1974), Abs. No. 76474a. In addition, several patents relate to glucose isomerization of which U.S. Pat. Nos. 3,616,221; Re. 28,885 (originally 3,623,953); 3,694,314; 3,708,397; 3,715,276; 3,788,945; 3,826,714; 3,843,442; 3,909,354; 3,960,663; 4,144,127; and, 4,308,349 are representative.
The levels of fructose achievable by the isomerization of glucose with glucose isomerase is limited by the equilibrium of the isomerization reaction. At 65xc2x0 C., the equilibrium of the reaction has been reported to stand at approximately 51% fructose by weight from a starting substrate of pure dextrose. Under standard conditions, the conversion of glucose to fructose is generally done at 60xc2x0 C. to 75xc2x0 C. and at a pH between 7 and 9. In this case, normally only 42% of fructose is obtained because of the equilibrium between glucose and fructose. To shift this equilibrium towards fructose, the temperature must be increased. However, most of the commercial glucose isomerases work at neutral to high pHs and the isomerization at high temperature and pH generates the formation of secondary reactions and undesirable bitter sub-products such D-psicose (Hiromichi, I. Rugh, S. et al. 1995, J. Ferm. Bioeng. 80: 101-103).
To attain syrups of higher fructose content, fractionation systems must be employed which add greatly to the cost of the final product. At higher temperatures, however, the equilibrium becomes more favorable. For example, an enzymatic glucose isomerase process capable of being operated at temperatures of from about 90xc2x0 C. to 140xc2x0 C. could be used to directly provide high fructose corn syrups (HFCS) containing 53-60 weight percent fructose on a dry basis, thereby eliminating the need for fractionation and recycle. The tendency of known glucose isomerase systems to undergo thermal denaturation with an accompanying sharp reduction in activity has thus far frustrated attempts to utilize higher temperature regimes to force the equilibrium of the isomerization further in favor of fructose. Moreover, glucose and especially fructose are sensitive reducing sugars which have a marked tendency to form unwanted by-products such as psicose, colored products, color precursors, fructose dianhydrides, mannose, tagatose, and acids when heated to the temperatures necessary to isomerize.
Several thermostable glucose isomerases have been described, e.g. from Thermus thermophilus (Dekker K., Sugiura A., et al., 1992, Appl. Microbiol. Biotechnol. 36 727-732); Thermotoga maritima (Brown S H. et al., 1993, Biotechn. Bioeng. 41: 878-886); Bacillus sp. (Wuxiang, L. and Jeyaseelan, K., 1993, Biotechn. Lett. 15: 1101-1106) and Streptomyces rubiginosus (Wong, H. C. et al., 1991, J. Bacteriol. 173: 6849-6858) and Chlostridium thermosaccharolyticum (Menden P G., Opoku , J A., Reizer J, Reizer A et. al, 1994; Gene: 141: 97-101). Nevertheless, the optimal pH of these thermostable glucose isomerases is generally over neutrality.
Thus, presently, there are several glucose isomerases having a high temperature for optimal functionality, such as glucose isomerases studied on the basis of Streptomyces flavovirens, Streptomyces olivochromogenes, Streptomyces violaceoniger, Lactobacillus brevis. However, all of these enzymes have a pH optimum which is relatively high (7.5 to 9). Karima Srih-Belghith and Samir Bejar (1998) Biotechnology Letters, Vol 20, No 6, June 1998, pp. 553-556, which is incorporated herein by reference.
It is an object of the present invention to provide improved polypeptides having glucose isomerase activity and nucleic acid encoding the polypeptides.
It is also an object of the present invention to provide a polypeptide having glucose isomerase activity with increased specific activity on glucose, fructose, xylose, and/or xylulose compared to other commercially available glucose isomerases.
The present invention relates to isolated polypeptides having glucose isomerase activity selected from the group consisting of:
(a) a polypeptide having an amino acid sequence which has at least 95% identity with amino acids of SEQ ID NO:2;
(b) a variant of the polypeptide having an amino acid sequence of SEQ ID NO:2 comprising a substitution, deletion, and/or insertion of one or more amino acids;
(c) a fragment of (a) that has glucose isomerase activity; and
(d) a polypeptide having a pH optimum in the range of 5.7 to 6.3 at 60xc2x0 C., a pH optimum in the range of 6.1 to 6.7 at 90xc2x0 C. and a temperature optimum of above 90xc2x0 C.
The present invention also relates to isolated nucleic acid sequences encoding the polypeptides and to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides.